PROJECT SUMMARY Sepsis is a clinical syndrome, which complicates severe infection. It remains the leading cause of morbidity and mortality in critically ill patients. Current concepts suggest that organ failure and mortality in sepsis are caused by inappropriate regulation of host defenses and the immune system. This manifests as an inability to control bacterial growth and dissemination, and excessive inflammation, processes that are interrelated and are due, in a large part, to macrophage dysfunction. Danger-associated molecular patterns (DAMP)s comprise a diverse group of molecules that accumulate in the extracellular space in response to bacteria-mediated tissue destruction, trauma, and burns, all of which are associated with sepsis. ATP is a major DAMP, which is released from the intracellular into the extracellular space through connexins and pannexins during inflammation, infection, shock, and sepsis. Extracellular ATP binds to and signals through P2 receptors to modulate immune function. Our preliminary data with mice deficient in P2 receptors, as well as pharmacological receptor and channel ligands establish that ATP release through connexin/pannexin channels and subsequent P2 receptor activation are crucial for the control of mortality, bacterial killing and dissemination and inflammation following cecal ligation and puncture, a clinically relevant model of polymicrobial sepsis. Mechanistically, we show that ATP and one of the P2 receptors, the P2X4 receptor, protects against sepsis by directly increasing the killing of bacteria by macrophages. Based on these data, we hypothesize that ATP governs the host's response to sepsis by signaling through P2 receptors on macrophages. To address this hypothesis, we will pursue the following Specific Aims: 1. Identify the mechanisms that support ATP release during sepsis. 2. Dissect the mechanisms by which P2X4 receptor stimulation augments the killing of sepsis- causing extracellular bacteria by macrophages. 3. Identify the monocyte/macrophage populations that are targets of P2 receptor-mediated immune regulation in sepsis. The overarching goal here is to delineate the role of P2 receptors in protecting against severe infection and to determine whether these receptors can be specifically targeted to manage patients with sepsis.